| BackgroundBreast cancer has a high morbidity and mortality rate.There are about 300,000new cases of breast cancer in China every year,and nearly 30%of them die from it.Breast cancer ranks the first among tumors that threaten woman's health.Breast cancer also has a high incidence of metastasis,among which bone tissue is a common site of metastasis.In the early stage of breast cancer,the incidence of bone metastasis is about 25%,while in the middle and late stage of breast cancer,the incidence of bone metastasis is as high as 80%,and once the occurrence of bone metastasis,nearly80%of patients will not reach the five-year survival period.At the same time,bone metastases also lead to serious bone-related adverse events,resulting in an extreme decline in the quality of life of patients.Therefore,we should seek treatment for bone metastasis of breast cancer.According to the molecular classification of breast cancer,it is found that different types of breast cancer cells have different tendency of organ metastasis,among which triple-negative breast cancer is more prone to brain metastasis and lung metastasis,while luminal breast cancer has a higher rate of bone metastasis.According to the above characteristics,through analyzing the clinical data of breast cancer in TCGA database,we found that the expression level of complement activation product C5a and its receptor C5aR2 were significantly increased in luminal breast cancer compared with other types.Complement protein is involved in tumor-related immune responses,as well as the formation of tumor micro-environment.When tumors appear in the body,the inflammatory response follows,in which the innate immune system is first activated.As an important part of the innate immune system,the activation of complement system is the first.In the process of complement activation,a large number of complement activation products will be produced,among which anaphylatoxin can spread to various parts of the body and play a role,and the intensity and influence of C5a in anaphylatoxin is the greatest.Therefore,the regulatory role and mechanism of complement activation product C5a and its receptors in breast cancer bone metastasis is the focus of this study.Objective(1)To clarify the correlation between complement activation product C5a/C5aRs and bone metastasis of breast cancer.(2)To investigate the role of complement activation product C5a/C5aRs in regulating the micro-environment of bone metastasis in breast cancer.(3)To explore the molecular mechanism of complement activation product C5a/C5aRs regulating bone metastasis in breast cancer.Method(1)Firstly,through the analysis of TCGA database,breast cancer-related clinical data were analyzed,focusing on the gene expression of luminal breast cancer related to bone metastasis.The correlation between complement activation product C5a and its receptors and bone metastasis was further analyzed by bioinformatics.Subsequently,the expression levels of complement activation product C5a and its receptors were detected by Q-PCR using cell lines with bone metastasis tendency and samples with bone metastasis in breast cancer.In this study,the expression level of C5a and its receptors was determined by IHC and ELISA.In order to further confirm the correlation between complement activation product C5a/C5aRs system and bone metastasis,we used sh RAN interference to knock down the C5aR1,C5aR2 of breast cancer cells respectively.A bone metastasis model was built by left ventricle injection breast cancer cells to observe the occurrence of bone metastases and changes of bone immune micro-environment.(2)By building an animal model of pre-metastasis micro-environment of breast cancer,the effect of C5a/C5aRs pathway on micro-environment formation before bone metastasis of breast cancer was observed.The role of C5a/C5aRs pathway in regulating bone metastasis in breast cancer was observed through in vitro experiments.Firstly,the effect of C5a/C5aRs pathway on breast cancer cell migration was observed by Transwell experiment,and the effect of C5a/C5aRs pathway on breast cancer cell proliferation was detected by clone formation experiment.The role of C5a/C5aRs pathway in regulating bone micro-environment was clarified through osteoblast differentiation,osteoclast differentiation and recruitment of osteoclast precursor cells.The effect of C5a/C5aRs pathway on immune micro-environment was also examined by flow cytometry.The effect of C5a/C5aRs pathway on regulating bone micro-environment was confirmed by the method of CRISPR-Cas9 gene editing after knocking out complement activation product C5a receptors.(3)In terms of mechanism research,firstly,C5a was used to induce the knockout of C5aR1 or C5aR2 breast cancer cell lines,and the conditioned medium was collected to explore the ability of cells to respond to C5a induction through osteoclast differentiation function.The differentially expressed proteins were analyzed by protein micro-array,and the signal pathways with more obvious effects were identified after KEGG enrichment.Then,the signal pathway was detected by osteoclast differentiation experiment by adding target signal pathway related protein inhibitors.The results were confirmed by Western Blot.From the results of protein micro-array analysis,different proteins,OPN and PD-L1 related to bone micro-environment and immune micro-environment were screened out.In the experiments to verify the results of the protein chip,the expression levels of OPN and PD-L1 genes were detected by Q-PCR.Finally,the protein chip was verified at the protein level through the use of OPN and PD-L1 neutralizing antibodies.Results(1)By analyzing TCGA database,we found that complement activation product C5a/C5aR2 pathway was highly expressed in luminal breast cancer.Bioinformatics analysis showed that breast cancer patients with high expression of complement activation product C5a and its receptors were more prone to bone metastasis,and compared with primary focus of breast cancer,complement activation product C5a receptors were also highly expressed in breast cancer bone metastases focus.In bone metastasis prone cell lines and breast cancer bone metastasis specimens,high expression levels of complement activation product C5a and its receptors were also detected.It was also found that the level of complement activation product C5a in serum of patients with bone metastasis of breast cancer was higher than that of in situ cancer.Immunohistochemistry showed that the expression of complement activation product C5a receptors in bone metastasis of breast cancer was higher than that in primary breast cancer.In vivo experiments,the incidence of bone metastasis in breast cancer cells knocked down C5a receptors decreased,and the number of MDSCs in bone marrow immune micro-environment decreased.(2)By adding complement activation product C5a directly and conditioned medium induced by complement activation product C5a,the regulation of C5a/C5aRs system on pre-metastasis micro-environment of breast cancer was studied.The results showed that C5a/C5aRs system could promote the migration and proliferation of breast cancer cells directly,and it could promote the recruitment of osteoclast precursor cells and the differentiation of osteoblasts and osteoclasts in regulating bone micro-environment.Moreover,complement activation product C5a/C5aRs system could also inhibit CD4~+T lymphocytes and recruit more MDSCs.(3)After complement activation product C5a induced knockout of C5aR1 or C5aR2 breast cancer cells,compared with the control group,the function of the conditioned medium to promote osteoclast differentiation was reduced.Through the analysis of protein micro-array,PI3K-AKT and MAPK signaling pathways were the most obvious changes after KEGG enrichment.After adding the protein inhibitors related to the above signaling pathways,the function of conditioned medium induced by complement activation product C5a in inducing osteoclast differentiation was as follows:different types of ERK inhibitors interfered the function of conditioned medium in inducing osteoclast differentiation.Western blot assay also showed that complement activation product C5a could regulate ERK phosphorylation in breast cancer cells.By analyzing the differentially regulated proteins of complement activation product C5a induced breast cancer cells,it was found that expression of OPN and PD-L1 were significantly different in bone micro-environment and immune micro-environment.After induction with complement activation product C5a,the expression of OPN and PD-L1 at m RAN level increased in a dose-dependent manner,while the expression of OPN and PD-L1 at m RAN level decreased after knocking down C5aR1 or C5aR2 and adding ERK inhibitor.In this study,after adding OPN neutralizing antibody,it was found that the function of conditioned medium of breast cancer cells in promoting osteoblast differentiation was weakened,and after adding PD-L1 neutralizing antibody,the function of conditioned medium in inhibiting CD4~+T lymphocyte was also weakened.In addition,the combination of the two neutralizing antibodies showed that the function of recruiting MDSCs by conditioned medium was further inhibited than that by adding alone.Conclusion:This study showed that complement activation products C5a and C5aRs were closely related to bone metastasis of breast cancer.In the process of bone metastasis of breast cancer,C5a and C5aRs are involved in regulating the formation of the micro-environment related to bone metastasis of breast cancer,promoting the differentiation of osteoblasts and osteoclasts in the bone micro-environment,and promoting the migration and proliferation of tumor cells.In the bone marrow immune micro-environment,the C5a/C5aRs pathway also plays an immunosuppressive role.Related mechanism studies showed that C5a combined with C5aRs regulated the expression of PD-L1 and OPN through the ERK signaling pathway,playing an important role in the bone marrow micro-environment. |
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